The Binding of Oxidized Low Density Lipoprotein (ox-LDL) to ox-LDL Receptor-1 Reduces the Intracellular Concentration of Nitric Oxide in Endothelial Cells through an Increased Production of Superoxide

doi:10.1074/jbc.M010612200

The Binding of Oxidized Low Density Lipoprotein (ox-LDL) to ox-LDL Receptor-1 Reduces the Intracellular Concentration of Nitric Oxide in Endothelial Cells through an Increased Production of Superoxide*

From the Department of Biomedical and Surgical Sciences, Verona University, 37134 Verona, Italy and ^§Department of Bioscience, National Cardiovascular Center Research Institute, Fujishirodai, Suita, Osaka, 565-8565, Japan

Abstract

Oxidized low density lipoprotein (ox-LDL) has been suggested to affect endothelium-dependent vascular tone through a decreased biological activity of endothelium-derived nitric oxide (NO). Oxidative inactivation of NO is regarded as an important cause of its decreased biological activity, and in this context superoxide (O⨪₂) is known to inactivate NO in a chemical reaction during which peroxynitrite is formed. In this study we examined the effect of ox-LDL on the intracellular NO concentration in bovine aortic endothelial cells and whether this effect is influenced by ox-LDL binding to the endothelial receptor lectin-like ox-LDL receptor-1 (LOX-1) through the formation of reactive oxygen species and in particular of O⨪₂. ox-LDL induced a significant dose-dependent decrease in intracellular NO concentration both in basal and stimulated conditions after less than 1 min of incubation with bovine aortic endothelial cells (p < 0.01). In the same experimental conditions ox-LDL also induced O⨪₂ generation (p < 0.001). In the presence of radical scavengers and anti-LOX-1 monoclonal antibody, O⨪₂formation induced by ox-LDL was reduced (p < 0.001) with a contemporary rise in intracellular NO concentration (p < 0.001). ox-LDL did not significantly modify the ability of endothelial nitric oxide synthase to metabolizel-arginine to l-citrulline. The results of this study show that one of the pathophysiological consequences of ox-LDL binding to LOX-1 may be the inactivation of NO through an increased cellular production of O⨪₂.

Footnotes

↵* This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; the Organization for Pharmaceutical Safety and Research; Takeda Science Foundation; and DNO Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Dipartimento di Scienze Biomediche e Chirurgiche, c/o Medicina D, Ospedale Policlinico, Università di Verona, 37134 Verona, Italy. Tel.: 39-045-8074806; Fax: 39-045-583041; E-mail: comina@medicinad.univr.it.
Published, JBC Papers in Press, January 24, 2001, DOI 10.1074/jbc.M010612200
Abbreviations:

NO

nitric oxide

O⨪₂

superoxide

ox-LDL

oxidized low density lipoprotein

LOX-1

lectin-like ox-LDL receptor-1

BAEC(s)

bovine aortic endothelial cell(s)

ROS

reactive oxygen species

MDA-LDL

malondialdehyde-modified LDL

CHO

Chinese hamster ovary

BLOX-1

bovine LOX-1

HE

hydroethidine

n-LDL

native LDL

Ac-LDL

acetyl-LDL

TB4

tetrahydrobiopterin

l-NMMA

l-N-monomethyl arginine

l-NAME

l-N-arginine methyl ester

DPI

diphenyleneiodonium

eNOS

endothelial nitric oxide synthase

MFI

mean fluorescence intensity

d-NMMA

d-N-monomethyl arginine

DAF-2 DA

4,5 diaminofluorescein diacetate
- Received November 26, 2000.
- Revision received January 18, 2001.
The American Society for Biochemistry and Molecular Biology, Inc.